The present invention relates to a specific and sensitive immunochromatographic (xe2x80x9cICTxe2x80x9d) assay, performable within about 15 minutes, for the detection of Streptococcus pneumoniae in a bodily fluid, such as urine or cerebrospinal fluid, of a patient showing clinical signs of an infection caused by S. pneumoniae. 
Streptococcus pneumoniae (xe2x80x9cS. pneumoniaexe2x80x9d) is a leading causative organism of pneumonia-type illnesses and other lower respiratory tract infections such as bronchitis, as well as of upper respiratory tract infections, including infectious otitis media and sinusitis and of disseminated invasive infections, including bacteremia and meningitis. When not properly diagnosed and treated, S. pneumoniae pneumonic infection may lead to any of pericarditis, empyema, purpura fulmiran, endocarditis or at least one type of arthritis, where S. pneumoniae is the causative organism in each instance. Such pneumonic infection is also often a precursor of bacteremia or meningitis. To now, it nevertheless is common for pneumonia arising from S. pneumoniae to be diagnosed and treated somewhat empirically.
To a significant extent, this is because the tests presently available for the detection of S. pneumoniae are either (1) time consuming, labor intensive and in need of instrumental assistance for reading results, or (2) lacking in sensitivity and/or specificity. Because of problems associated with lack of sensitivity and/or specificity, e.g., physicians tend toward conservatively prescribing expensive, broad spectrum antibiotics for patients with pneumonia-type respiratory infections in lieu of prescribing a less expensive antibiotic specific to S. pneumoniae where it would adequately cure the infection. This and other liberal prescribing of broad spectrum antibiotics is, of course, a major cause of today""s well-publicized medical crisis consequent from the increasing resistance of many types of infectious bacteria to previously highly efficacious antibiotics. This crisis and the potential untoward consequences for at least some patients of empirical diagnosis and treatment are among many reasons why a reliable and rapid assay for detecting S. pneumoniae in human body fluids is needed.
Pneumonia caused by S. pneumoniae is a serious disease, estimated to occur at the rate of one to five cases per 1,000 persons per year in the United States alone. Depending upon the age and state of health (based on unrelated factors) of patients infected with S. pneumoniae-caused pneumonia, the disease has a mortality rate of between 4 percent and 30 percent of infected patients.
The most time-honored methods of attempting to diagnose S. pneumoniae-caused diseases, and especially pneumonia, involve the Gram stain and culture of expectorated sputum of patients suspected of harboring the disease, followed by biochemical identification methods. This procedure requires in the order of one to four days from start to finish. It has proved to be an unsatisfactory diagnostic tool because (1) other bacteria present in the patient""s saliva often overgrow the sputum culture, and (2) S. pneumoniae frequently is present in the human upper respiratory tract even when no sign of disease attributable to this bacterium is present in the individual. For example, it is estimated that some 30 percent of U.S. children are habitual carriers of S. pneumoniae. Adults, too may become colonized by S. pneumoniae without themselves entering a disease state. The carriage rates of the organism by both children and adults increase with crowding conditions and during winter months.
Co-agglutination, latex particle agglutination and counter-immunoelectrophoresis methods for detecting the polysaccharide capsular antigens of S. pneumoniae in sputum specimens have been developed and are rapid, but they have not been shown to exhibit reliable sensitivity or specificity, probably because there are some 83 serotypes of S. pneunoniae, each of which may vary in immunogenicity and in other respects. The commercial polyvalent anti-serum developed and used for these tests contains antibodies to all 83 of the S. pneumoniae serotype antigens, but it nevertheless may fail to detect the less immunogenic antigen serotypes. This polyvalent antiserum also has shown cross-reactivity with other streptococci and some other infectious bacteria, e.g., Haemophilus influenzae. Hence both false-negative and false-positive reactions may occur randomly when these tests are used on sputum samples.
Several enzyme-inmmunoassays (xe2x80x9cEIAxe2x80x9d) have been developed which are based on detection of the pneumococcal C-polysaccharide antigen that has been found to be present in the pneumococcal cell wall of all of the S. pneumoniae serotypes. See, e.g., Parkinson, A. J., Rabiego, M. E., Sepulveda, C., Davidson, M. and Johnson, C., 30 J. Clin. Microbiol. 318-322 (1992). This C-polysaccharide antigen is a phosphocholine-containing polysaccharide derived from teichoic acid. These EIA assays are of acceptable specificity and sensitivity even though most often performed on sputum samples. Each such assay, however, requires two to three hours performance time after sample collection as well as the use of instrumentation normally available primarily in clinical laboratories. In addition, these assays need to be run by, or under close supervision of, trained personnel.
Reliance upon sputum samples to diagnose S. pneumoniae infections is frequently less than satisfactory in achieving a diagnosis of S. pneumoniae-caused pneumonia, and not just because of the potential for contamination of the sample by other bacteria in the mouth and/or by indigenous upper respiratory tract S. pneumoniae. Sputum is often difficult to collect; moreover, once medication of the patient is commenced, the number of viable S. pneumoniae in sputum rapidly decreases. In particular, the presence of the C-polysaccharide antigen in sputum may rapidly become difficult to detect if an antibiotic therapy is used that attacks the cell wall of the S. pneumoniae microorganism. When S. pneumoniae causes infectious otitis media, meningitis and various other aforementioned infectious disease states, sputum samples are of no aid in diagnosis.
Collection of blood cultures from patients suspected of S. pneumoniae infection eliminates the contamination problems that attend sputum samples. Where blood serum samples are found to contain S. pneumoniae, diagnosis of various diseases of which it is causative may readily be made. The drawback here is that only about 20 percent of all pneumonia patients infected by S. pneumoniae become bacteremic; therefore, relying solely on blood samples to diagnose S. pneumoniae-caused pneumonia may yield false-negative results.
Urine samples have been found to be the most reliable and convenient ones to use in detecting S. pneumoniae-caused pneumonia because they can be non-invasively obtained; they will not be contaminated with oral microflora; and the presence of the bacterium in urine persists, albeit at a constantly decreasing level of concentration, even after patient therapy has been initiated, so that daily monitoring of patient urine samples to assess the efficacy of a prescribed therapy may yield useful information. It should be noted that human carriers of S. pneumoniae who show no disease symptoms often do not have sufficient pathogen present to have S. pneumoniae antigens present in their urine.
A very recent article describes the successful diagnosis of meningitis caused by S. pneumoniae using an EIA method to test samples of cerebrospinal fluid. In the EIA, a monoclonal immunoglobulin A antiphosphoryl-choline antibody was employed to detect the C-polysaccharide antigen. See Stuertz, K., Merx, I., Eiffert, H., Schmutzhard, E., Mader, M. and Nau, R., 36 J. Clin. Microbiol. 2346-2348. The results obtained compared favorably with those reported by Yolken, R. H., Davis, D., Winkelstein, J., Russell, H. and Sippel, J. E., 20 J. Clin. Microbiol. 802-805 (1984) obtained in an EIA in which two antibodies for S. pneumoniae in cerebrospinal fluid were usedxe2x80x94a horse antibody to the pneumococcal C-polysaccharide antigen, bound to microtiter plates, and a pooled rabbit antiserum to the polysaccharide capsular antigen in the liquid phase.
According to the present invention, antibodies to the C-polysaccharide antigen of S. pneumoniae raised in rabbits are affinity purified with isolated and purified C-polysaccharide antigen having less than about 10% protein content.
These affinity purified antibodies are conjugated to an agent which produces a color reaction upon the formation of a sandwich with S. pneumoniae C-polysaccharide antigen from a test sample and additional affinity purified C-polysaccharide antibody immobilized upon a nitrocellulose matrix.
The test is conducted in a disposable immnunochromatographic test device and requires no instrumentation to interpret the result. It can easily and successfully be performed by persons who have no training in laboratory techniques.
The preferred test sample for diagnosis of S. pneumoniae-caused pneumonia is patient urine, but the test also works with other bodily fluid samples that contain S. pneumoniae, including serum and sputum. Diagnosis of S. pneunoniae-caused meningitis may be readily made using patient cerebrospinal fluid as the test sample.
This invention for the first time offers the benefit of a test for S. pneumoniae that is performable within a 15-minute time span and is of at least equal specificity and sensitivity to EIA tests requiring eight to twelve times as long and much more work, to obtain a result. The test is easy to perform, requires no special training, equipment, or instrumentation and it enables a rapid diagnosis of pneumonia caused by S. pneumoniae. It can be readily performed in a doctor""s office, thus permitting the patient to be immediately placed on a S. pneumoniae-specific therapeutic regimen. It can, of course, be performed in a clinical laboratory, but it can also easily be performed in a geriatric center, in a patient""s home or in any environment where S. pneumoniae-caused pneumonia or other pathogenic condition is suspected to be epidemic.
The test of this invention is important to administer when disease states such as otitis media, bronchitis or sinusitis appear because once it can be established that any of these is due to S. pneumoniae rather than another infectious agent, appropriate therapy can promptly be initiated. Small children are especially prone to otitis media because of the shorter length and smaller diameter of their Eustachian tubes, so that early detection of S. pneumoniae if present may well forestall the onset of a more serious, or even life-threatening, disease state. Papers by Norris et al, J. Pediatrics, 821-827 (1966) and Hongeng et al, 130 J. Pediatrics, No. 5 (May 1997) indicate that children with sickle cell disease are highly susceptible to S. pneumoniae infection, with S. pneumoniae sepsis being the most common invasive infection among this populace and those once so infected having a much heightened risk of recurrence and subsequent death. Clearly, employing the ICT test of this invention to test the urine of these patients on a regular basis may be helpful in diminishing the need for the unremitting penicillin prophylaxis that the second of these papers recommends.
The ease of performance of the test and its ability to detect the C-polysaccharide antigen of S. pneumoniae in urine suggests that this test should prudently be performed on patients without overt clinical signs of related infection who report feeling substantially under par. Any such patient in whom it is established that S. pneumoniae is present in significant enough quantities to give a positive-urine ICT test is a predictable candidate for developing a more severe infectionxe2x80x94and the ability to forestall the disease development before it becomes severe by administering appropriate therapy is newly presented by this invention.